Structural Destabilization of Azurin by Imidazolium Chloride Ionic Liquids in Aqueous Solution

Abstract

Alkyl imidazolium chloride ionic liquids (ILs) have been used for numerous biochemical applications. Their hydrophobicity can be tuned by changing the alkyl chain length, and longer-chain ILs can form micelles in aqueous solution. We have investigated the effects of imidazolium chloride ILs on the structure and stability of azurin, which is a very stable Cu2+ redox protein with both α-helix and β-sheet domains. Temperature-dependent infrared (IR) and vibrational circular dichroism spectroscopy can provide secondary-structure-specific information about how the protein is affected, and temperature-jump transient IR measurements can quantify the IL-influenced unfolding dynamics. Using these techniques, we can quantify how azurin is destabilized by 1.0 M ILs in aqueous solution. The shorter, less hydrophobic ILs, 1-butyl-3-methylimidazolium chloride and 1-hexyl-3-methylimidazolium chloride likely interact with the α-helix domain and decrease protein melting temperature from 82 °C without IL to 55 °C and disturb the overall tertiary structure, resulting in a looser, more open shape. Thermodynamic analysis indicates that protein destabilization is due to increased unfolding entropy. 1-Octyl-3-methylimidazolium chloride [OMIM]­Cl, which forms micelles in solution that may partially solvate the protein, has a more significant destabilizing effect, resulting in a melting temperature of 35 °C, larger unfolding entropy, and relaxation kinetics several orders of magnitude faster than with unperturbed azurin. The temperature-independence of the relaxation time constant suggests that in the presence of [OMIM]­Cl, the protein folding potential energy surface has become very smooth.

Effects of Ionic Liquids on Metalloproteins

“…A brief summary of studies that have been published on azurin with ILs can be found in Table 4. [Cl] is stronger than the other ILs presented in this study, which were consisting of smaller alkyl chains and a decreased level of hydrophobicity [87]. It is important to note that at the concentrations tested, the [OMIM][Cl] has been shown to form micelles, These micelle structures likely impact the interactions with the protein, and can potentially form mixed structures with the protein.…”

“…Standard Fourier transform IR (FTIR) can monitor secondary structures in the protein, while far-infrared spectroscopy (FIR) can be utilized to interrogate low-frequency vibrations, such as those in metal complexes [85,86]. These IR methods have been used to investigate the structure of numerous metalloproteins and peptides, including EndoIII, azurin, bovine serum albumin, and natural and designed peptides [86][87][88][89][90][91]. All of these pieces of information can together provide information regarding the stability of a protein's structure [92].…”

“…The blue copper protein, azurin, is part of the azurin-nitrate reductase redox protein complex. This protein is involved in denitrification metabolism in bacteria [87,179]. The presence of copper is necessary for protein stability.…”

“…It is a small protein that can be produced from two bacterial strains-Pseudomonas aeruginosa and Alcaligenes denitrificans [180]. Azurin's structure from P. aeruginosa consists of a hydrophobic alpha helix, six short beta sheets and a random-coil that allows for copper-binding [87,181,182] (Figure 3). Notably, azurin exhibits the most blue-shifted Trp emission spectrum from naturally derived proteins, arising from the single Trp residue at position 48 [183].…”

“…It is important to note that at the concentrations tested, the [OMIM][Cl] has been shown to form micelles, These micelle structures likely impact the interactions with the protein, and can potentially form mixed structures with the protein. [87].…”